The effects of the Wnt/β-catenin signaling pathway on the in vitro differentiation of rat BMSCs into leydig cells

Abstract

Late-onset hypogonadism (LOH) refers to sexual and non-sexual symptoms in men caused by age-related decreases in circulating testosterone. Leydig cells (LCs) transplantation is considered to be one of a viable approach for LOH therapy, but the limited source of LCs limits the application of this approach. The aim of this study was to induce the directed differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into LCs in vitro, and explore the potential involvement of Wnt/β-catenin signaling pathway in the differentiation process. BMSCs were extracted from rats and characterized by flow cytometry for positive rates of mesenchymal stem cell markers CD29, CD44, CD90, and the hematopoietic marker CD45. BMSCs were divided into three groups: Control, Wnt agonist (CHIR-99021), and Wnt inhibitor (LGK-974), each incubated for 14 days. ELISA and RT-qPCR were used to verify the protein and mRNA expression of β-catenin, LRP5 and TCF, the key factors in Wnt/β-catenin signaling pathway. The average fluorescence intensity of 3β-hydroxysteroid dehydrogenase (3β-HSD) on the surface of LCs was detected by immunofluorescence (IF) assay. The content of testosterone secreted in cell culture medium was detected by ELISA. The results of flow cytometry indicated that we successfully extracted and cultured BMSCs. Moreover, post 14 days of incubation, the changes of β-catenin, LRP5 and TCF, at the protein and mRNA level demonstrate successful intervention in the activation and inhibition of the intracellular Wnt/β-catenin signaling pathway. Compared with the control group, the LCs surface marker 3β-HSD expression intensity in the CHIR-99,021 group was significantly increased by 69% (p < 0.01), while significantly decreased by 59% in LGK-974 group (p < 0.01). The ELISA results indicated a higher testosterone concentration in the CHIR-99,021 group (359.58 ± 17.46 pg/mL) than in the control (225.31 ± 15.42 pg/mL) and LGK-974 groups (183.67 ± 4.47 pg/mL), and the difference was statistically significant (p < 0.05). This study successfully demonstrates the directed differentiation of BMSCs into LCs under the action of inducers. We verified that the Wnt/β-catenin signaling pathway is involved in this differentiation process. The idea proposed in our study for efficiently inducing differentiation of BMSCs into LC in vitro, may provide a safe and sustainable LC source for developing clinically feasible cell transplantation-based LOH therapies.

Keywords: Bone mesenchymal stem cell; Late-onset hypogonadism; Leydig cell; Testosterone; Wnt/β-catenin.

Fig. 1

Schematic diagram of the mechanism of Wnt/β-catenin signaling pathway and the targets of CHIR-99,021 and LGK-974.

Fig. 2

The expression of BMSCs surface markers CD29, CD45, CD90 and CD44. (A) The set gate and collected cell scatter diagrams. (B) PE-H-CD45 antibody. (C) APC-H-CD29 antibody. (D) FITC-H-CD44 antibody. (E) Pacific Blue-A-CD90 antibody.

Fig. 3

Cell morphology of BMSCs in different groups and relative expression levels of LRP5, β-catenin, TCF in each group. (A) a: Rat BMSCs (200×); b: Control group (200×); c: CHIR-99,021 group (200×); d: LGK-974 group (200×). (B) The protein expression levels of LRP5, β-catenin, and TCF in control group, CHIR-99,021 group and LGK-974 group. (C) The mRNA expression levels of LRP5, β-catenin, and TCF in three different groups of cells. (*, p < 0.05 vs. control group; **, p < 0.01 vs. control group)

Fig. 4

3β-HSD immunofluorescence staining of testicular LCs and the content of testosterone in culture medium. (A) The expression of 3β-HSD in control group, CHIR-99,021 group and LGK-974 group. The green fluorescence was positive expression of 3β-HSD, and the DAPI staining was blue fluorescence with nuclear staining (1000×). (B) The relative mean fluorescence intensity of 3β-HSD in each group. (C) The testosterone secretion levels in three groups of cells. (*, p < 0.05 vs. control group; **, p < 0.01 vs. control group)